Of these, higher levels of miR-1, miR-495 and miR-4454 could be validated by real-time PCR. In addition, elevated miR-4454 levels were significantly correlated with cardiac fibrosis, detected by magnetic resonance imaging in HCM patients. (4) Conclusions: Circulating miR-1, miR-495-3p and miR-4454 levels are elevated in the plasma of HCM patients. To the best of our knowledge, this is the first report showing a correlation between miR-4454 levels and cardiac fibrosis in HCM. This suggests miR-4454 as a potential biomarker for fibrosis in these patients.(1) Background: Left ventricular hypertrophy, myocardial disarray and interstitial fibrosis are the hallmarks of hypertrophic cardiomyopathy (HCM). Access to the myocardium for diagnostic purposes is limited. Circulating biomolecules reflecting the myocardial disease processes could improve the early detection of HCM. Circulating miRNAs have been found to reflect disease processes in several cardiovascular diseases. (2) Methods: We quantified circulating miRNA molecules in the plasma of 24 HCM and 11 healthy controls using the Human v3 miRNA Expression Assay Kit Code set  and validated differentially expressed miRNAs using RT-PCR. (3) Results: In comparison to healthy controls, the levels of six miRNAs  were higher in the plasma of HCM patients than healthy individuals ( Hypertrophic Cardiomyopathy (HCM) is a common inherited cardiac condition with a prevalence of 1:200 to 1:500 in the general population . It is tA decade ago, several groups succeeded in detecting circulating miRNAs in plasma and serum, raising the question of the potential of miRNAs as biomarkers for various diseases ,6,7,8,9.Therefore, the purpose of this study was to determine potentially relevant miRNA molecules as biomarkers for HCM in a hypothesis-free approach. We analyzed circulating miRNAs using the nCounter Sprint Digital Analyzer  in patients with HCM and controls and related differentially expressed miRNAs to cardiac phenotypes. This technique is based on a direct fluorescent staining of the miRNA molecules and therefore enables the quantification of the authentic levels of all tested circulating miRNAs without the amplification of previously selected target molecules.A total of 24 patients with HCM and 11 healthy controls were recruited in the University Heart Center Hamburg between May 2010 and August 2015. All patients were outpatients at the time of blood sampling. The clinical study protocol was approved by the local ethics board and written informed consent was obtained from all patients and controls. The diagnosis of HCM was based on the current criteria of the European Society of Cardiology guidelines  and cel-miR-254  were added to control for variances in starting material and RNA-extraction efficiency. RNA isolation was performed according to the manufacturer’s protocol. Due to very low RNA yields, all samples were concentrated using RNA Clean & Concentrator-5 Columns . RNA Quality Control was performed using the NanoDrop 2000C Spectrophotometer . 8/µL Qiagen cat. No. MSY0000010) was added to 200 µL of plasma. The isolation was carried out according to the supplementary protocol for the miRNA Easy Kit (Qiagen Purification of RNA from serum or plasma (RY43 Feb-11). This kit combines phenol/guanidine-based lysis of plasma samples and column-based extraction to purify total RNA. Extracted RNA was eluated in 30 µL of RNase-free water. For RT-PCR, RNA was isolated using the miRNA Easy Kit  using 200 µL of plasma of each study participant. According to the manufacturer’s instructions, 3.5 µL of diluted syn-spike-in-miR-cel-39 (1.6 × 10Plasma expression levels of 800 different miRNAs were quantified using the Human v3 miRNA Expression Assay Kit Code set (#CSO-MIR3-12) and nCounter Sprint Digital Analyzer  according to the manufacturer’s conditions. This new sensitive method allows the direct digital detection of miRNA without reverse transcription or amplification. The technology is based on direct molecular barcoding of target molecules through ligation of sequence-specific, color-coded probe pairs to the miRNAs. The probe pair consists of a 3′biotin-labeled capture probe and a 5′fluorescent-labeled reporter probe. Following hybridization and enzymatic purification, the miRNA-probe pair constructs were immobilized on a cartridge for data collection in the nCounter platform . Backgro® LNA® RT Kit). UniSp6 RNA spike-in was added to the master mix as instructed. Three microliters of cDNA (diluted 1:30) templates were used for the subsequent real-time PCR. Appropriate miRCURY LNA miRNA PCR Assays  for the candidate miRNAs were used. CT values of more than 35 were excluded from the analysis. Relative quantification normalized to miR-cel-39 was done using the ΔΔ-Ct method. Results are given in fold change. To validate the results of the top-6 candidate miRNAs, RT-qPCR was performed with the miRCURY LNA miRNA PCR Kit  on a Quant Studio 6 Flex Real-Time Cycler according to the supplied protocol. Four microliters of eluted RNA were used for RT  were determined using Welch’s T-test. Pearson correlation analysis and linear regression between miRNA levels and clinical parameters were calculated using Prism version 8.2.1. Statistical significance was considered if The analysis of echocardiographic data showed significant thickening of the septal (23 ± 6 vs. 10 ± 1 mm) and inferolateral wall (16 ± 4 vs. 9 ± 1 mm) in HCM patients compared to controls . Furtherp < 0.05) higher in plasma from HCM patients than from the controls. No miRNAs with lower plasma concentrations in HCM patients were detected. The miR-3144 exhibited a 2.3-fold higher level in HCM patients than in the control individuals , followed by a 2.1-fold higher level of miR-495-3p (154 ± 30 vs. 74 ± 9) and a 2-fold higher level of miR-627-3p in HCM patients than in the controls  were significantly (p = 0.02) and miR-4454 level was 2.1-fold higher in HCM patients (2.10 ± 0.35 vs. 1.00 ± 0.12). The other three miRNAs could not be detected in the plasma of the patients by RT-qPCR.All miRNAs showing differential expression on the exploratory screen were quantified by RT-PCR. Of the six putative miRNAs that were identified with the NanoString technology, three were validated by RT-qPCR . The miRp = 0.001) and of septal wall-thickness    with clinical parameters was carried out. The level of miRNA-4454, but not of miR-1 and miR-495-3p was correlated with the extent of myocardial fibrosis detected by LGE on MRI  . Based o 0.2358)  in HCM pIn this hypothesis-generating study, we could identify and validate three miRNAs being regulated in the plasma of HCM patients using NanoString nCounter as a new screening approach. This is the first study in which miRNA-4454 could be identified as being associated with HCM, and it shows a correlation between miRNA-4454 and hypertrophy and fibrosis in the entire cohort. This study evaluated the expression pattern of circulating miRNAs in patients with HCM compared to healthy controls using the innovative nCounter platform . This technique allows for the direct digital measurement of isolated miRNAs by a sequence-specific hybridization of every single miRNA molecule with a unique fluorescence code . Compared to RT-qPCR, which is considered to be the gold standard of miRNA measurement, the miRNA Expression Assay from Nanostring Technologies does not require reverse-transcription and amplification steps and therefore reflects authentic miRNA molecule counts in the plasma samples. However, sample input per run is limited to a small sample number with the nCounter platform. Therefore, this method is not feasible for high throughput screening. The v3 Human miRNA Expression Assay Code Set Kit provides tags for 800 miRNAs including all published high confidence and clinical miRNAs based on the miRbase, as well as numerous positive and negative controls making it a convenient, easy-to-use, and time-saving miRNA detection method for approaches with small populations, e.g., rare diseases. Several circulating miRNAs are known to be differentially regulated in patients with HCM , some of which, especially miR-29a, are also suggested to be associated with myocardial fibrosis, a hallmark of HCM ,19,20,21In our cohort, we found six circulating miRNAs  with significantly higher levels in HCM patients than in the healthy controls. For three of them , an impact on cardiac diseases was already described ,23. Luo Thus far, two studies show downregulation of miR-1 in cardiac tissue of patients with HCM ,26. HoweEnhanced levels of three other miRNAs  in HCM patients were detected, none of which were described to have an impact on cardiovascular diseases so far. The miR-627 level was higher in gastric cancer, activated by calcitriol and suppressed CYP3A4 in colon cancer cells and was described to modulate TGF-β1-induced pulmonary fibrosis ,33,34. MIn our study, of these six miRNAs only miR-1, miR-495-3p and miR-4454 could be validated with RT-qPCR, probably due to the low expression of the other miRNAs in human plasma as previously proposed . All thrSince cardiac fibrosis has been associated with cardiac arrythmias, finding a specific marker for cardiac fibrosis is of particular interest . BesidesThis study has limitations. First, the number of HCM patients was small. Second, since circulating miRNAs were analyzed in patient plasma, the miRNA origin and their potential role in cardiac diseases remain to be determined in future studies. Therefore, our results should be considered as a hypothesis-generating study that needs to be validated in larger cohorts. All presented miRNA levels were measured in plasma samples. The origins of these circulating miRNA molecules are unknown and can only be conjectured based on tissue-specific expression .With the innovative nCounter digital analyzer, we identified six miRNAs that exhibit higher levels in the plasma of HCM patients than in controls. Three of them were validated by RT-qPCR. In our cohort, miRNA-4454 directly correlated with the septal LV wall thickness and the extent of fibrosis. Further, miRNA-4454 correlates with the extent of fibrosis in HCM patients. These results suggest that miR-4454 might serve as a potential biomarker for myocardial fibrosis in hypertrophic cardiomyopathy.


Ironically, the presentation of a subset of studied material as retrieval cues at test often impairs recall of the remaining (target) material—an effect known as part-list cuing impairment. Part-list cues are typically provided at the beginning of the recall period, a time when nearly all individuals would be able to recall at least some studied items on their own. Across two experiments, we examined the effects of part-list cuing when student participants could decide on their own when the cues were presented during the recall period. Results showed that participants activated the cues relatively late in the recall period, when recall was already close to asymptote. Critically, such delayed cuing no longer impaired recall performance. The detrimental effect of part-list cuing, as it has been demonstrated numerous times in the memory literature, thus seems to depend on presentating the cue items (too) early in the recall period. Bothlaboratory and applied memory research has demonstrated that episodic remembering can benefit enormously from the presence of adequate retrieval cues. Recall of a categorized list can be improved if the items’ category names are provided as retrieval cues at test . They were equally distributed across the three between-participants conditions, resulting in n = 48 participants in each condition. We determined the desired sample size based on reported effect sizes in prior part-list cuing work from our lab . Participants in the standard part-list cuing condition were provided the 12 cue items at the beginning of the recall period, whereas participants in the self-paced part-list cuing condition could activate the same 12 cue items anytime during the recall period. In the no-part-list cuing condition, participants were not provided with any part-list cues. Assignments to part-list cuing conditions, lists, and list sets were counterbalanced across participants.In the study phase, the items of a list were exposed on a computer screen for 3.5 s per item and in a random order. The study phase was followed by a 2-min distractor phase, in which participants solved arithmetic problems as a recency control. At test, all participants received a sheet of paper with the unique initial letters of the 12 target items, presented in two columns of six items each and in a random order. Participants were asked to fill in the missing target items in any order they wished for the next 2 min. Prior to this 2-min recall period, participants in the standard part-list cuing condition were provided the 12 cue items in three columns of four items each on the computer screen. The order of the items was random. Participants were told to use these items as retrieval cues for recall of the list’s target items. The cue items were presented sequentially at a 2 s rate and participants were asked to read each item aloud. Presented items remained on the screen until the end of the recall period. The self-paced part-list cuing condition was identical to the standard part-list cuing condition, with the only difference that participants could determine when during the recall period (and if) the 12 cue items were provided. Participants were told that they could push a button on the computer keyboard, which initiated presentation of the list’s remaining 12 items on the computer screen; these items could serve as retrieval cues for recall of the target items and would stay present on the screen for the rest of the recall period. Cue presentation time (12*2 s) was discounted from duration of the recall period. In the no-part-list cuing condition, no cue items were presented.%CI = ). A rough breakdown of activation times shows that 15 participants activated the cues within the first 30 s, 32 participants within the first 60 s, and 40 participants within the first 90 s  showed a significant effect of condition, F = 5.08, MSE = 387.15, p = .007, η2 = 0.07. Follow-up comparisons revealed that standard part-list cuing impaired target recall relative to the no-part-list cuing condition , t(94) = 2.91, p = .005, d = 0.59, whereas target recall in the self-paced part-list cuing condition did not differ from recall in the no-part-list cuing condition , t(94) < 1, d = 0.13. Consistently, target recall in the self-paced condition was significantly higher than in the standard condition, t(94) = 2.63, p = .010, d = 0.54.Figure As expected, most participants in the self-paced part-list cuing condition did not activate the part-list cues particularly early in the recall period, which contrasts with the standard part-list cuing condition, in which the cue items were provided at the beginning of the recall period. This difference in the timing of part-list cuing was accompanied by a marked difference in recall performance between the two cuing conditions, with higher recall in the self-paced than the standard part-list cuing condition. Moreover, recall in the self-paced condition was similar to recall in the no-part-list cuing condition, indicating that the typical detrimental effect is eliminated when cuing is self-paced. Together, the findings on cue activation times and recall levels suggest that participants in the self-paced condition activate the cue items sufficiently late in the recall period to shield (most of) the target items from the potentially detrimental effects of blocking, inhibition, or strategy disruption. The goal of Experiment M= 22.17 years, range, 18-38 years, 72.9% female, 27.1% male). Sample size followed Experiment n = 48 participants in each condition. All participants spoke German as native language and received monetary reward or course credit for participation.Another 144 students of Regensburg University took part in the experiment  for the first cue package, 78.24 s  for the second cue package, and 88.56 s  for the third cue package. A rough breakdown of activation times shows that 14 participants activated the first cue package within the first 30 s, 34 participants within the first 60 s, and 44 participants within the first 90 s  = 3.77, MSE = 411.02, p = .026, η2 = 0.05. Follow-up comparisons revealed that standard part-list cuing impaired target recall compared to the no-part-list cuing condition , t(94) = 2.21, p = .030, d = 0.45, whereas self-paced part-list cuing left recall largely unaffected relative to the no-part-list cuing condition , t(94) < 1, d = 0.03. Again, target recall in the self-paced condition was significantly higher than in the standard condition, t(94) = 2.62, p = .010, d = 0.54.Figure Like in Experiment This view on self-paced part-list cuing suggests that recall in Experiments M= 22.23 years, range, 18-28 years, 83.3% female, 16.7% male). Sample size followed Experiment Another 48 students of Regensburg University took part in the experiment  = N(1 − et/τ−), where F(t) represents the cumulative number of items recalled by time t, N represents asympotic recall, i.e., the estimated percentage of items that could be produced given unlimited time, and τ represents mean response latency to recall those N items  after 30 s, 43.06%  after 60 s, 46.70%  after 90 s, and 49.13%  after 120 s. The observed recall level after 120 s was nearly identical to the recall level observed in the no-part-list cuing condition of Experiment M = 49.48%, 95%CI = ), t(94) < 1, d = 0.02. Figure τ and 48.71 for parameter N, which is close to the recall level observed after 120 s and indicates that participants would not have been able to recall many more items if the recall period had been prolonged.Cumulative target recall was 33.85%  material is often impaired. This study shows that this core finding does not generalize to self-paced part-list cuing, when individuals can decide on their own when during the recall period the cues are provided. The potentially detrimental effects of cues on recall may thus be circumvented with self-paced cuing.


A serum sLOX-1 concentration was measured by the enzyme-linked immunosorbent assay (ELISA). Results: The results obtained showed a statistically significant raised level of sLOX-1 in pre/post PCI patients of stable CAD/ACS with male preponderance. The area under the curve for sLOX-1 was 0.925 for cases that are discriminated from controls with sensitivity and specificity of 87.88 and 100%, respectively. SLOX-1 showed 100% sensitivity and specificity in the discrimination of the stable CAD that underwent PCI vs. control with an AUC of 1.00. The recurrence of coronary artery disease was observed in 9 out of 132 (6.8%) cases. The post-interventional sLOX-1 level was significantly different and higher in recurrent cases (p = 0.027) of ACS/CAD. Conclusions: sLOX-1 was a useful biomarker of stable CAD/ACS and has a potential in the risk prediction of a future recurrence of coronary artery disease.Background: Cardiac troponin is the best marker to diagnose acute coronary syndrome (ACS). However, early diagnosis using markers for plaque instability may be of significance. Soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) plays an important role in the pathogenesis of atherosclerosis plaque rupture and may be a potential biomarker of coronary artery disease (CAD), including ACS. The current study aims to evaluate sLOX-1 levels in the sera of patients with ACS as an independent marker of CAD with other established diagnostic markers and assess its level before and after percutaneous intervention (PCI) in predicting the risk of future recurrence of ACS. Methods: Peripheral blood was obtained from a total of 160 patients, including patients who underwent coronary angiography ( Cardiovascular disease remains a major cause of mortality and debility globally, of which coronary artery disease (CAD) accounts for the highest percentage , althougLectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the receptor for atherogenic oxidized low-density lipoprotein (Ox-LDL). The pro-inflammatory stimuli and ox-LDL induce the expression of LOX-1, which affects the atherosclerotic progression and plaque vulnerability ,4. SolubStudy subjects were recruited between May 2018 and June 2019 from the Lari Cardiology Centre, Department of Cardiology, King George Medical University, Lucknow, UP, India. All the cases that were recruited during this study period were included to constitute the sample size. Patients undergoing angiography and diagnosed with ACS and stable CAD (pre-PCI and post PCI) were included in the study. All angiographically proven subjects showing variable degrees of cardiac vessel blockage were categorized as cases, and the rest of the subjects as well as first degree relatives of these patients from the healthy population were categorized as control, and both of the groups were evaluated further by biochemical and hematological examination and follow-up for one year. All the patients who fulfilled the inclusion criteria were recruited for the study. Written informed consent was taken from the patients. The research related to human use has been complied with institutional policies and follows the tenets of the Declaration of Helsinki. The study was approved by the Institutional Ethical Committee of King George’s Medical University, Lucknow (ID 95th ECM II B-T/P13). Informed consent was obtained from all subjects involved in the study.g for 10 min and stored at −80 °C until further processing.A total of 3.5 mL of peripheral blood was collected from cases and control subjects in plain and EDTA vials . The serum was separated by centrifugation at 1900× All biochemical parameters, including serum thyroid stimulating hormone (TSH), 25-hydroxy vitamin D, vitamin B12, folate, and homocysteine (HCY), were measured by a fully automated biochemical analyzer . Selectra 500 Pro XL was used to measure magnesium and creatine kinase-MB (CK-MB) levels according to the manufacturer’s instructions. The neutrophil lymphocyte ratio (NLR) was calculated by taking the absolute count of neutrophils divided by the total count of lymphocytes. The platelet lymphocyte ratio (PLR) was calculated as the absolute count of platelets divided by the absolute count of lymphocytes. Complete blood count investigations involved measuring all differential values on Swelab Alfa Automated Hematology Analyzer .The serum was separated by centrifugation (1900 g for 10 min) and stored at −80 °C for the quantification of sLOX-1 concentrations. The serum sLOX-1 was quantified by a commercially available enzyme-linked immunosorbent assay (ELISA) kit as per the manufacturer’s instructions . The kit was with an intra-assay coefficient of variation of <10% and an interassay CV of <12%. ELISA routinely analyzed all the samples in triplicate, and the results were averaged to minimize measurement errors and calculate the final concentration in samples. The level of the sLOX-1 level was measured in terms of ng/mL. We compared the diagnostic value of the sLOX-1 level of the different groups vs. the control group.After a primary PCI, all patients were monitored for at least 24 h. The patients were given standard medications by consulting physicians according to the up-to-date guidelines. After discharge, all patients were followed up for one year in an outpatient setting. Electronic patient records identified the recurrence of all patients. The follow-up started on the day of admission, and the patients were prospectively followed for 1 year with a standard medical treatment. All the patients were followed up completely. The recurrence of CAD was associated with recurrent cardiovascular events, including stroke and recurrent myocardial infarction (MI).We analyzed the sLOX-1 as a diagnostic indicator across the spectrum of acute coronary syndrome, but a number of tables are related with recurrence or treatment outcome scenarios.p-value < 0.05 (two-tailed) was considered significant.The statistical analysis was performed using SPSS  Version 21.0 statistical analysis software. Normally distributed continuous variables were presented as mean ±SD (standard deviation) and in number (%). The ANOVA test followed by post-hoc tests (Tukey’s HSD) was used to compare the within-group and between-group variances. A receiver operating characteristic curve (ROC) was developed to evaluate the diagnostic performance of sLOX-1. 2 × 2 tables built categorizing tests as positive or negative based on individual cut-offs obtained from ROC curves. A n = 18), who underwent coronary angiography, but did not have established CAD; group II: patients of stable CAD underwent percutaneous intervention (n = 50); group III: patients of the acute coronary syndrome (n = 64); and group IV: healthy controls (n = 28). The age of study subjects ranged from 23 to 85 years of age. The mean age of patients enrolled in the study was 47.94 ± 14.30 years. The mean age of group I was 49.33 ± 15.66 years, that of group II was 46.40 ± 14.82 years, that of group III was 48.78 ± 14.36 years, and that of group IV was 47.89 ± 12.76 years. In the current study, the baseline characteristics, including age, gender, diabetes mellitus, and lipid profiles, were comparable and did not differ significantly between the groups. We aim to investigate and compare the pre/post PCI levels of sLOX-1 and other biochemical markers in stable ACS/CAD patients, followed up to one year to assess the future recurrence of the disease, and the sLOX-1 and other biochemical markers were measured in the predictor of recurrence of ACS/CAD.A total of 160 subjects, including 132 cases and 28 controls, were included in the study. The study subjects were divided into 4 groups: group I: patients underwent coronary angiography (p < 0.001). Similarly, the vitamin D level was significantly higher in the control group (group IV with 31.84 ± 6.44 ng/mL), and group II was 16.29 ± 3.99 ng/mL, when compared to group I and III (p < 0.001). The CK-MB levels in group II patients (67.78 ± 73.22 IU/L) were significantly higher than group I, group III, and controls (group IV). The HCY level of group II (41.75 ± 45.10 μmol/L) was significantly higher than the levels of the other groups. For the groups least followed during the pre-treatment, the sLOX-1 level of group IV was 28.68 ± 9.97 units, whilst that of groups I, II, and III was 44.43 ± 42.86 units, 815.04 ± 683.38 units, and 995.16 ± 611.77 units, respectively (p < 0.001). The sLOX-1 levels of group I and group IV were comparable, while group II and group III had significantly higher sLOX-1 levels than group IV.The pre-treatment serological parameters  for all the study groups were compared and depicted in p < 0.001). The pre-treatment sLOX-1 level was significantly higher between group I vs. group II, group I vs. group III, and group II vs. group IV (p < 0.001). The folate level was significantly different between group I vs. group IV and group II vs. group IV (p < 0.001). The analysis of the group differences reveals that the CK-MB level was significantly different between group I vs. group II, group II vs. group III, and group II vs. group IV (p < 0.001).The comparisons of pre-treatment serological parameters between groups are shown in p < 0.001), CK-MB levels in group II , and HCY levels in group II ; and a subsequent rise in the post-intervention sLOX-1 level of groups I, II, and III  was observed. The maximum change in pre-treatment sLOX-1 levels was observed for group III (95.50%), followed by that in group I (66.57%); in group II, the change was kept to a minimum (39.55%) while rest parameters did not differ significantly.The post-intervention serological parameters in the different groups are depicted in p < 0.001).The comparisons of post-interventional serological parameters between groups are shown in A pre-treatment sLOX-1 ROC curve analysis was performed at a cut-off with a higher value that indicated the need for coronary angiography . The arep = 0.655). However, the post-interventional sLOX-1 level was significantly different and higher in recurrence cases (p = 0.027). Other pre- and post-interventional serological parameters did not show any association with recurrence.The recurrence of CAD was associated with recurrent cardiovascular events, including stroke and recurrent myocardial infarction (MI). A recurrence of coronary artery disease was observed in 9 out of 132 (6.8%) cases. A recurrence of coronary artery disease was observed in 4.0% of group II and 10.9% of group III; however, no recurrence was observed in group I. The recurrence rate of CAD among the three groups was not statistically significant, as depicted in The current study compares the utility of sLOX-1 levels and other biochemical markers in the risk assessment of patients of acute coronary syndromes/CAD for future recurrence/morbidity/mortality. Our study showed a statistically significant increase in the pre-treatment levels of sLOX-1 in all the studied groups, with the order of increase being: patients with ACS > patients with stable CAD > patients who only underwent coronary angiography > controls. There was a statistically significant rise in the post-treatment sLOX-1 levels in all the patient groups except controls, with the order of increase being: patients with ACS > patients with stable CAD > patients who only underwent coronary angiography.We have not performed a multivariate analysis and primarily focused on the independent use of the sLOX-1 level for prediction. We plan to perform a multivariate assessment in a separate study to see the association of sLOX-1 levels with different clinical and demographic conditions. Such multivariate assessment is out of the context of the present study and beyond its scope. Given the high sensitivity and specificity of the sLOX-1 level for predicting various coronary artery conditions, a multivariate assessment was not considered essential.p = 0.027). In the present study, pre-treatment CK-MB levels in stable CAD patients were statistically higher than those in other groups. Post-interventional increases in CK-MB levels were found to be statistically significant for all three patient groups. A study by Ullah et al. reported that elevated cardiac biomarkers after PCI might be associated with worse clinical outcomes. This also helps to identify high-risk post PCI groups [In coherence with our results, Hayashida et al. found sLOX-1 as a useful marker for the early diagnosis of ACS . A studyI groups . HoweverIn our study, pre-treatment HCY levels in stable CAD patients were significantly higher than those in other groups. A post-treatment increase in HCY levels was found to be statistically significant for all three patient groups. In a study by Rajabi et al., the mean serum homocysteine of diabetic patients (type II) with CAD was higher than those with normal coronary arteries and was significantly higher in patients with three-vessel involvement than those with normal coronary arteries that had no vessel involvement . We haveThere were no significant differences in serum magnesium levels in intergroup comparison of pre- or post-treatment levels. Qin et al. reported that serum levels of vitamin B12, vitamin D, and vitamin C were in close correlation to the vascular endothelial dysfunction in CAD patients . In our We have comparatively analyzed the sLOX-1 level as an independent diagnostic marker of ACS with few established markers and tried to assess whether its levels before and after PCI may predict the risk of a future recurrence. Consistent with the earlier studies, we have found increased sLOX-1 levels in all the patient groups with a statistically significant increase in all the groups before and after PCI. We have also found a significant correlation of post-treatment sLOX-1 level with a future recurrence. The level of sLOX-1 is associated with the incidence of periprocedural myocardial infarction . Our finIn our study, nutritional and hormonal status showed inconsistent results. There was no significant increase or decrease in these entities’ pre- or post-PCI levels, except for low folate levels, which showed significant association with CAD/ACS, thus warranting further investigation in larger studies from an Indian perspective. Considering the very short duration of this study, it is recommended to conduct a multicentric study with long-term follow-up to achieve a better outcome. For instance, this study has limitations of a small-sized observational study. Risk factors, including smoking, diabetes, and body mass index (BMI), which were different in the groups, were not analyzed and may have affected the results of the biomarkers studied.


Madurella mycetomatis. Treatment of mycetoma consists of a combination of a long term antifungal treatment with itraconazole and surgery. However, treatment is associated with low success rates. Therefore, there is a need to identify novel treatments for mycetoma. CIN-102 is a synthetic partial copy of cinnamon oils with activity against many pathogenic bacteria and fungi. In this study we determined the in vitro activity of CIN-102 against 21 M. mycetomatis isolates and its in vivo efficacy in a M. mycetomatis infected Galleria mellonella larval model. In vitro, CIN-102 was active against M. mycetomatis with MICs ranging from 32 μg/mL to 512 μg/mL. 128 μg/mL was needed to inhibit the growth in 50% of tested isolates. In vivo, concentrations below the MIC of 40 mg/kg and 80 mg/kg CIN-102 prolonged larval survival, but higher concentrations of CIN-102 did not.Mycetoma is a devastating neglected tropical infection of the subcutaneous tissue and most commonly caused by the fungus  Madurella mycetomatis. Mycetoma is treated with antifungal agents and surgery but with low success rates. In our search for novel treatments for mycetoma we determined if CIN-102, a synthetic oil that resembles cinnamon oil, would be therapeutic in mycetoma. We therefore determined the activity of this oil against the fungus in vitro. We found that CIN-102 could inhibit M. mycetomatis growth in vitro. To determine if CIN-102 was also active against the mycetoma grain in vivo, we determined the efficacy in our Galleria mellonella grain model. Interestingly, CIN-102 was found to be effective in the larvae at a low concentrations.Mycetoma is a tropical infection causing large tumorous lesions on mainly the foot. This infection is most commonly caused by the fungus  Madurella mycetomatis is the most common causative agent world-wide -2H-tetrazolium hydroxide (XTT) assay [The antifungal activity of CIN-102 against T) assay . In shorM. mycetomatis MM55. To obtain a hyphal suspension, MM55 was cultured on SDA at 37°C for three weeks. The mycelium was harvested, sonicated for 30 seconds at 28 micron  and incubated in RPMI 1640 medium containing 0.35 g/L L-glutamine, 1.98 mM 4-morpholinepropanesulfonic acid (MOPS) and 100 mg/L chloramphenicol at 37°C for two weeks. To obtain the fungal mycelia, the culture was filtered through a 22 μm filter (Whatman), the biomass was scraped from the filter, weighed, re-suspended in PBS and sonicated for 2 minutes at 28 microns. Followed by centrifugation at 3400 rpm for 5 minutes, the biomass was adjusted with PBS to a concentration of 1 g/10 mL. Larvae were injected with 40 μl of inoculum via the last left pro-leg, resulting in a final concentration of 4 mg wet weight of fungi per larvae. Larvae were divided into groups of 15 and each group was treated on three consecutive days at 4, 28 and 52 hours post-infection. Administered dosages consisted of either 40 mg/kg, 80 mg/kg, 400 mg/kg or 800 mg/kg CIN-102 solved in 10% tween-80 in PBS. Additionally, 5.71mg/kg itraconazole  and solvent only (10% tween-80 in PBS) control groups were included [In order to determine the toxicity and efficacy of CIN-102, 45 larvae/concentration were injected with CIN-102 via the left last proleg and monitored for 10 days. The tested CIN-102 concentrations were 800 mg/kg, 400 mg/kg, 80 mg/kg and 40 mg/kg solved in 10% tween-80 in PBS. To determine the efficacy of CIN-102, 15 larvae/concentration were infected with a hyphal suspension of the genome isolate 2), medium (0.01–0.019 mm2) and small (0.005–0.009 mm2) sizes as described by Lim et al. [To determine the fungal burden within the larvae, larvae were infected and treated as described above. At 24 and 72 hours post-infection, five larvae from each group were sacrificed for histological examination and assessment of fungal burden. The larvae were fixed in 10% buffered formalin for 24 hours, dissected longitudinally into two halves with a scalpel and fixed for another 48 hours before further routine histological processing . The twom et al. . The summ et al. .To compare the survival lines and determine if there was a statistical difference between the different treatment groups, the Log-rank test was performed with GraphPad Prism 8 . A Mann-Whitney test was performed to determine if the number of grains or the size of the grains within the larvae differed between larvae treated with CIN-102, ITZ or solvent. A p-value smaller than 0.05 was deemed significant.As shown in As can be seen in To assess the fungal burden within the larvae, the number and the size of the grains were determined by histological examination 24 and 72 hours after fungal infection. As shown in Although treatment with CIN-102 did not result in a difference in the size and number of grains, differences were observed regarding the physiology surrounding the fungal grain. After treatment with 80 mg/kg CIN-102, encapsulation occurred earlier and a decrease in the cement material was observed compared to the controls. Similar findings were observed for itraconazole. However, unique to treatment with CIN-102 was the observation of hemocytes with a larger morphology surrounding the grain. These were not present in the control group or the itraconazole treated group .M. mycetomatis growth with MICs ranging from 32 μg/mL  to 512 μg/mL . A concentration of 256 μg/mL  was needed to inhibit the growth of 90% (MIC90) of the isolates. This was comparable to the activities found of different essential oils generated from Cinnamomum spp. against other fungal species where for most of these essential oils, 40 μg/mL to 310 μg/mL was needed to inhibit the growth of Candida spp., Cryptococcus neoformans, Trichophyton spp., Microsporum spp. and Aspergillus fumigatus determined by broth microdilution following CLSI guidelines [Candida spp. and Aspergillus spp. growth, respectively [In this experimental work we explored the possibility of the synthetically well defined, synergistic blend CIN-102 as a potential novel therapeutic for mycetoma. As demonstrated in this study, CIN-102 was able to inhibit idelines . The maiectively .M. mycetomatis infected larvae. Within these larvae, characteristic mycetoma grains are formed. Furthermore, the antifungal efficacy studies performed in this model appeared to be predictive of the efficacy studies performed in mice [in vitro data. Furthermore, despite the enhanced survival, no reduction in the total number of grains or the size of the grains was noted. The observation of earlier encapsulation of the grains and enlarged hemocytes in the larvae suggested a possible influence of CIN-102 on the G. mellonella immune response. A limitation of the current study is that we did not investigate the immune reaction of the G. mellonella larvae towards CIN-102. However, some clues can be obtained from literature. Cinnamaldehyde, the major compound of CIN-102, has been described to up-regulate expression and production of anti-inflammatory cytokines IL-10 and TGF-β, while down-regulating pro-inflammatory cytokine expression and production [CIN-102 was able to prolong larval survival in  in mice ,28. Inteoduction . CIN-102oduction –33. It woduction .G. mellonella larvae was not only due to the immediate fungicidal effect of CIN-102 to the fungus but the combination of fungicidal activity and an altered immune response of the larvae towards the mycetoma grain.The importance of the host immune system in treatment outcome was recently demonstrated by the remarkable cure of a mycetoma patient upon the addition of non-steroidal anti-inflammatory drug (NSAID) diclofenac to the treatment alongside posaconazole and 5-flucytosine . It is tin vitro potency against M. mycetomatis and in vivo efficacy was demonstrated within M. mycetomatis infected Galleria mellonella larvae. This study shows the potential of chemically well-defined blends, which offer an interesting alternative to current single-drugs screening approaches. As mentioned earlier, this study was not designed to evaluate the immunological response upon treatment. Following this major limitation, more studies are required to determine the mechanism of action of CIN-102, the potential of other synergistic blends and the involvement of the immune response during fungal mycetoma infections.In conclusion, CIN-102 showed promising S1 FigIn vivo toxicity (panel A) and efficacy (panel B) of different dosages CIN-102 and the used treatment dosage itraconazole within G. mellonella larvae, compared to the solvent (PBS 10% tween80). In panel A, A concentration of 800 mg/kg CIN-102 is significantly different from the solvent control (***p = 0.0003), none of the other dosages effect the survival of uninfected larvae. In panel B, the dotted line indicates uninfected larvae as toxicity controls, which were treated at the same time points as the infected groups. The full lines indicate infected larvae treated with the corresponding dosages 4-, 28- and 52-hours post-infection. Larvae treated with solvent and itraconazole survive up until day 8, while larvae treated with both 40 mg/kg and 80 mg/kg survive up until day 10 and significantly differ from the PBS control (*p = 0.0133 and **p = 0.0091).(TIF)Click here for additional data file.


We studied the secondary attack rate (SAR), risk factors, and precautionary practices of household transmission in a prospective, longitudinal study. We further compared transmission between the Alpha (B.1.1.7) variant and non-Variant of Concern (non-VOC) viruses. From May 2020 throughout April 2021, we recruited 70 confirmed COVID-19 cases with 146 household contacts. Participants donated biological samples eight times over 6 weeks and answered questionnaires. SARS-CoV-2 infection was detected by real-time RT-PCR. Whole genome sequencing and droplet digital PCR were used to establish virus variant and viral load. SARS-CoV-2 transmission occurred in 60% of the households, and the overall SAR for household contacts was 50%. The SAR was significantly higher for the Alpha variant (78%) compared with non-VOC viruses (43%) and was associated with a higher viral load. SAR was higher in household contacts aged ≥40 years (69%) than in younger contacts (40–47%), and for contacts of primary cases with loss of taste/smell. Children had lower viral loads and were more often asymptomatic than adults. Sleeping separately from the primary case reduced the risk of transmission. In conclusion, we found substantial household transmission, particularly for the Alpha variant. Precautionary practices seem to reduce SAR, but preventing household transmission may become difficult with more contagious variants, depending on vaccine use and effectiveness. SARS-CoV-2, the virus that causes the respiratory disease COVID-19, was first detected in China in 2019 and spread rapidly throughout the world ,4. It isDuring the pandemic, various genetic variants have evolved from the original SARS-CoV-2 virus . Some ofThe majority of household transmission studies have described transmission of the SARS-CoV-2 variants dominating in the early phase of the pandemic or have not described the genetic variant(s). The first confirmed case of the Alpha variant in Norway was reported in December 2020, and from mid-February 2021 until July 2021 it was the dominant variant . Even thWe conducted a prospective longitudinal household study to investigate the SAR in Norwegian households, and to identify risk factors for transmission and preventative measures, using frequent testing and biological sampling, together with questionnaire data. Close follow-up and systematic data collection allowed for determination of the role of viral load in transmission. We used the droplet digital PCR (ddPCR) technique to quantify SARS-CoV-2 viral RNA due to its greater accuracy and precision compared to traditional quantitative PCR (rRT-PCR) ,16. MoreThe design of this prospective longitudinal study was based on the WHO Household Transmission Investigation protocol . From MaPrimary cases and their household contacts were identified by the municipalities’ infection control teams following a positive SARS-CoV-2 PCR test and were subsequently contacted by the study team. Households willing to participate were visited at home, and written informed consent was obtained from the participants and/or their guardians before study inclusion.The study was approved by the Regional Ethics Committee in Norway (#118354).According to the Norwegian COVID-19 regulations, isolation was mandatory for persons with confirmed COVID-19. Isolation should be implemented at home or in similar accommodation for at least 8–10 days after symptom debut (recommendations varied throughout the study period), lasting at least three days after symptom relief. Asymptomatic cases had to isolate for 10 days after their initial positive PCR-test. In isolation, positive cases were instructed to stay ≥2 m from other household members, use separate bathrooms, towels, and bedrooms if possible. Household contacts were instructed to quarantine in their homes, maintaining an increased distance to other adults in the household.The first home visit for inclusion and sampling was termed Day 0, and seven further home visits for sampling were performed during the following 6 weeks  .®EDTA-k2, Greiner Bio One, Kremsmünster, Austria) was collected once for each participant aged ≥18 years for blood typing. Saliva and blood for immunological analyses were also collected at Day 0, Day 7, Day 14, Day 28, Day 42, and Day 180 (outside the scope of this article).Oropharyngeal (OP) samples and neat saliva samples were gathered from eligible participants on each visit to test for SARS-CoV-2 by rRT-PCR. Health care workers collected OP samples using OP flocked swabs , in 3 mL UTM . Whole blood (VacuetteAll participants were asked to answer a questionnaire on Day 0 (Q-D0), to obtain information about the household in general, transmission risk factors, precautionary practices, clinical symptoms, and general health status. This questionnaire was adapted from the WHO protocol. The questions on behavioral risk factors in the Q-D0 related to the period up to 10 days prior to SARS-CoV-2 confirmation of the primary case, and precautionary practices after confirmation. An additional questionnaire (Q-DX) with questions on the suspected source of transmission, adherence to isolation/quarantine regulations, and a self-report of the severity of disease, was answered by participants at the home visit on Day 28/Day 42 or collected through phone interviews. In addition, a symptom diary adapted from the WHO protocol was completed daily from Day 0 to Day 28 by all participants.N-acetylcystein (10 g/L) and shaken for 30 min. Viral transport medium was added to saliva samples with insufficient volume before extraction. A semi-quantitative real-time reverse transcription polymerase chain reaction (rRT-PCR) was performed using the AgPath-ID One-step RT-PCR kit  with primers and probes targeting two SARS-CoV-2 RdRp gene targets, developed at Institut Pasteur, Paris, France, and shared in the WHO protocol inventory [All OP and saliva samples were tested for the presence of SARS-CoV-2 by rRT-PCR at the Norwegian Institute of Public Health (NIPH). RNA was extracted from samples (200 µL) using MagNaPure 96 DNA and Viral NA Small Volume kits , and eluted in 50 µL. Saliva samples with too much mucus were mixed 1:1 with sputum lysis buffer containing nventory . A 25 µLFor absolute quantification of viral load (SARS-CoV-2 RNA copies per µL eluate), ddPCR was performed on the saliva sample with the lowest Ct value for each participant, if sufficient material was available. The positive samples were further analyzed by droplet digital PCR (ddPCR) for absolute quantification. Results from the rRT-PCR analysis were evaluated to identify samples with high viral load that needed dilution to allow ddPCR quantification . The PCRThe positive samples were further analyzed by amplicon-based whole genome sequencing (WGS) of SARS-CoV-2 using the ARTIC-network nCoV-19 protocol v3 ,21 usingBlood groups ABO and RhD were determined for all participants aged ≥18 years old using the Bio-Rad ID-Microtyping system at the blood bank of Oslo University Hospital.Household contacts were defined as individuals aged ≥2 years who resided with the primary case. A household contact was considered a secondary case if they had a positive PCR test , and their symptom onset/PCR positive test  was within 14 days after T0 of the primary case. If a household contact had a T0 ≥2 days prior to T0 of the original primary case, the household contact was defined as an alternative primary case. If household members had the same T0, or ±1 day, they were re-defined as co-primary cases, unless the original primary case had a known source of infection outside of the household.Household size was defined as the number of people living in the household. Overcrowding was defined as (1) the number of rooms in the property being less than the number of persons living in the household and (2) the number of square meters was less than 25 per person [Clinical severity was based on the question: “How ill did you feel?” with the following 5 response categories: (1) “not ill”, (2) “not very ill”, (3) “moderately ill”, (4) “quite ill”, or (5) “seriously ill”, combined with the presence or absence of symptoms and/or dyspnea reported in the Day 0 questionnaire and symptom diaries. Dyspnea was considered a more severe symptom. Symptoms had to arise within 14 days after symptom onset, to ensure they were related to the SARS-CoV-2 infection. Severity was then defined according to response category and symptoms as follows: asymptomatic (category 1 or no reported symptoms), mild (categories 2–4 or symptoms without dyspnea), or moderate (category 5 or dyspnea).n = 7; 4 children and 3 adults), resulting in a total of 56 household contacts in the analysis (21 children and 35 adults).Duration of detectable SARS-CoV-2 was defined as the mean number of days a household contact was positive for laboratory confirmed SARS-CoV-2, i.e., the date of the last positive saliva test minus the date of the first positive saliva test according to the test regime . The date of the first positive sample was in some cases derived from the initial laboratory test taken through the municipality. Participants with any negative SARS-CoV-2 samples that had been diluted prior to rRT-PCR analysis were excluded from this analysis  of household contacts that were defined as confirmed cases . ClusterTo account for dependencies within households, a mixed-effect logistic regression model with a household-level random intercept was used to study the associations between potential risk factors for transmission and of infection among the household contacts. The multivariable models were adjusted for age and sex of the household contacts and of the primary cases, and household size. The analysis on associations between SARS-CoV-2 viral load (SARS-CoV-2 RNA copies/µL eluate measured by ddPCR) and symptoms was limited to the confirmed cases. For analyses with all cases, a mixed-effect logistic regression adjusted for age and sex was used, whereas for analyses only done on primary cases logistic regression was used. To study the association between genetic variant and viral load, a mixed-effect linear regression adjusted for age and sex was used. The mean duration of detectable SARS-CoV-2 was estimated for household contacts only, for children (<18 years) and adults (≥18 years), and cluster robust standard errors were used to calculate 95% confidence intervals. Primary cases were not included in this analysis as the majority were adults (due to the inclusion criteria of the study) and infection was likely detected later in the course of disease for these participants. To estimate the association between duration of detectable SARS-CoV-2 by rRT-PCR (in days) and age group, a mixed-effect linear regression was used.p-value of <0.05 was considered statistically significant (shown in bold in the tables).All analyses were performed in STATA/SE 15.0 . A We recruited 70 households, including 216 participants . Ninety The median household size was four, ranging from two to six people, and families with young children constituted 43% of the households . The houp = 0.09) (p < 0.01) . Children were SARS-CoV-2 rRT-PCR positive for a shorter time period than adults (mean number of days 11.3 (95% CI 7.6–15.1) and 16.4 (95% CI 13.5–19.3), respectively, p = 0.03). The time period did not differ according to viral variant (data not shown). No association between blood type and variant was found (data not shown).Of the 200 participants, 132 (66%) were infected. Fourteen percent of the confirmed cases were asymptomatic, while 43% had mild disease, and 42% had a moderate disease, based on their reported symptoms within 14 days of their first positive PCR sample. Few study participants were hospitalized, and all were discharged the following day. There were slightly more asymptomatic cases (22%) among the Alpha variant participants compared with participants with non-VOC viruses (9%), although the difference was not significant ( = 0.09) . Severitp = 0.04). For household contacts, SAR was 77.8% (95% CI 49.4–92.6) in households with the Alpha variant, compared with 42.5% (95% CI 28.7–57.7) in households with non-VOC viruses, resulting in a significantly higher adjusted odds ratio (OR) for secondary transmission in households with the Alpha variant (p = 0.03).Secondary transmission occurred in 60.0% of the households in the study (95% CI 47.4–71.4) . The SARn = 50). The median serial interval was similar for the Alpha variant  and non-VOC viruses . The overall median interval between symptom onset of the primary case and the first rRT-PCR-positive test of a household contact was 3 days , and this interval was similar for Alpha  and non-VOC viruses .The median interval from the date of the first positive SARS-CoV-2 test  and the Day 0 visit in the study was 3 days . A large proportion of the secondary cases (38.5%) were already infected at Day 0, while 61.5% of the secondary cases were detected during study follow-up. The overall median serial interval (the number of days between symptom onset of the primary case and of a household contact) was estimated to 4 days  nor sex of the primary case appeared to have an impact on SAR . NotablySecondary infection amongst children aged 2–17 years was similar for those aged 18–39 , while household contacts aged ≥40 years were more likely to be infected (69%) . The sexNo difference in clinical severity was observed between the Alpha variant and other strains among the primary cases . Howeverp < 0.001). There was a trend that higher viral load measured by ddPCR was associated with increased risk of secondary infection (adjusted OR 3.05 (95% CI 0.84–11.0), p = 0.089). Higher viral load was also associated with increased risk of loss of taste/smell (adjusted OR = 1.4 (95% CI 1.06–1.85), p = 0.02)  determined by ddPCR and the rRT-PCR Ct-values was strong  . However = 0.02) .10 and 2.48 log10 RNA copies/µL eluate, respectively, p = 0.006)  (p = 0.001).The viral load was significantly higher for the Alpha variant than for non-VOC viruses (mean 3.24 log= 0.006) A. We alsctively) B, irrespctively) C. The asNone of the contact behaviors between the primary case and the household contacts prior to confirmation of infection of the primary case were significantly associated with SAR . Neverthp = 0.048)  . All othThis prospective longitudinal household study with close follow-up and systematic sampling shows a high overall SAR (49.6%), confirming that households are an important site of transmission. The SAR of the Alpha variant (B.1.1.7 VOC) was significantly higher, at 77.8%, compared with 42.5% for the other non-VOC viruses dominating in Norway until Feb/March 2021. A significantly higher viral load was found in the saliva of participants with the Alpha variant compared to the non-VOC viruses, which may contribute to the increased transmissibility. Close contact behavior prior to confirmation of infection of the primary case tended to give a higher SAR. However, we showed that SAR was reduced if the primary case slept in a separate room or was isolated from the rest of the household after infection was confirmed.Our SAR-estimate of 42.5% for non-VOC viruses is higher than the household SAR found in other early reviews showing pooled SAR estimates around 17% ,28. HoweThe Alpha variant has been shown to be generally more transmissible than non-VOC viruses ,11 and oPrevious estimates of SAR in children and different age groups, have been conflicting ,39,40,41We used ddPCR to accurately assess SARS-CoV-2 viral load and to avoid potential inference from inhibitory substances which may influence the results when using rRT-PCR for quantification . PreviouWe also demonstrated both lower viral load and shorter duration of viral detection in children compared with adults, which in accordance with some studies ,44,45, wMost contact behavior such as kissing, appeared to slightly increase the odds of secondary transmission, although not significantly. We found that sleeping separately from the primary case after confirmation of infection prevented secondary infection, as shown previously . Other mVaccination campaigns against SARS-CoV-2 are anticipated to reduce household transmission. However, a recent study from the UK suggests that the vaccines may be less effective at reducing household transmission against currently circulating strains  than expected . It is pThe present study has several limitations. First, our sample size was small, which limited the comparison between factors associated with the Alpha variant and other non-VOC viruses, such as symptoms and severity. Further, the study was not initially designed to evaluate differences in SAR between variants, and the dominance of the variants differed during the study period. We can therefore not exclude that climate, people’s behavior, or other factors, could have influenced our results. Quarantine and isolation guidelines were similar throughout the whole study period; thus, we assume that this has not significantly influenced our results. We cannot exclude that some participants may have used antiviral or antipyretic drugs during the study. However, antiviral drugs are not commonly used in Norway outside of hospital settings. Finally, the age span of participants was limited, with few elderly individuals and mostly adult primary cases.In this prospective longitudinal household study, we found an overall SAR for household contacts of 50%. The SAR was considerably higher for the Alpha variant (78%) than for non-VOC viruses (43%). Furthermore, the viral load was higher for the Alpha variant which may explain its increased transmissibility. We also showed that age affects secondary infection, with higher SAR in household contacts older than 40 years. Loss of smell/taste in the primary case was associated with increased transmission. Implementation of precautionary measures after detection of the first SARS-CoV-2 case seems to reduce household transmission, in particular sleeping separately from the primary case. However, preventing transmission within a household will become increasingly difficult with the emergence of more contagious variants, depending on vaccine use and effectiveness.


The two fluids were incubated with Caco-2 cell monolayers grown in Transwell® inserts, and paracellular permeability was measured as transport of FITC-dextran. Non-significant decreases (p > 0.05) in paracellular permeability were found, which was attributed to the relatively low phenolic concentration in the solutions tested  as quercetin (200 µM) and one of its microbial-derived phenolic metabolites, 3,4-dihydroxyphenylacetic acid (200 µM), led to significant decreases (p < 0.05). The expression of tight junction (TJ) proteins  in Caco-2 cells after incubation with IDW and CDW was also determined. A slight increase in mRNA levels for occludin for both IDW and CDW fluids, albeit without statistical significance (p > 0.05), was observed. Analysis of the microbiome and microbial activity during wine colonic fermentation revealed relevant changes in the relative abundance of some families/genera  as well as in the microbial production of SCFA , all of which might affect paracellular permeability. Both direct and indirect (microbiota-mediated) mechanisms might be involved in the protective effects of (wine) polyphenols on intestinal barrier integrity. Overall, this paper reinforces (wine) polyphenols as a promising dietary strategy to improve gut functionality, although further studies are needed to evaluate the effect on the intestinal barrier under different conditions.This paper explores the effects of wine polyphenols on intestinal permeability in in vitro conditions. A red wine  was sequentially subjected to gastrointestinal and colonic digestion in the Dynamic Gastrointestinal Simulator (simgi The intestinal barrier is a dynamic structure separating the internal host from the gut lumen and is comprised of several items: mucus and epithelial layers, immunological (lymphocytes and innate immune cells) and humoral (defensins and immunoglobulin A (IgA) elements, and muscular and neurological elements . Of specDietary components such as fiber and polyphenols might counteract inflammation processes and intestinal dysfunction, with their biological action being mediated through the gut microbiota . PolypheWine is probably one of the foods that shows greater and more diverse phenolic content composition. Within wine phenolic compounds there are flavonoid compounds such as flavan-3-ols, flavonols, flavones, and anthocyanins (only in red wine), and non-flavonoid compounds such as hydroxybenzoic acids, hydroxycinnamic acids, stilbenes and hydrolyzable tannins. To the best of our knowledge, so far, only two studies have evaluated the effect of grape/wine polyphenols on intestinal permeability in vitro. Nunes et al.  reported® . For example, in the study by Freire et al.  were added to the apical side of the Transwell® inserts containing the Caco-2 cell monolayers. These plates were incubated at 37 °C for 4 h, simulating the time for which dietary compounds remain in the small intestine in in vivo conditions.Differentiated cells were incubated with the digested-wine fluids (IDW or CDW) and pure phenolic compounds in two independent assays. In a first experiment, 500 µL of sample  were added to cell monolayers and incubated for 16 h, which simulated the average time that dietary components remain in the colon. For both experiments, incubations were carried out in triplicate and experiments were repeated on three different days.In a second experiment, in a similar way, 500 µL of sample colonic digested wine (CDW) or the colon nutrient medium (CDM)  solution containing 1 mg/mL of fluorescein isothiocyanate (FITC-)-dextran 4 kDa and the basolateral sides of the plate inserts were filled with DPBS. Then, the plate was incubated at 37 °C for 30 min. Afterwards, 100 µL from the basolateral chamber of each well were taken in triplicate to measure the concentration of FITC-dextran by fluorescence intensity in a BioTek FL600 microplate reader  with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.For both experiments, when the incubation time was over, the apical chamber solutions of the Transwell® RNA XS kit  following the manufacturer’s instructions. cDNA was obtained with a qPCRBIO cDNA Synthesis Kit  according to the procedure described by the manufacturer.At the end of the experiments, Caco-2 cell monolayers were scraped and withdrawn with 500 µL of cold PBS, which was centrifuged at 1500 rpm for 10 min. Then, 350 µL of the RA-1 buffer (with 1% of β-mercaptoethanol) was added to the pellet prior to the mRNA extraction, which was carried out with a NucleoSpinFinally, qRT-PCR was performed employing a ViiA™ 7 Real-Time PCR System  to quantify the genetic expression of the TJ proteins ZO-1 and occludin using the GADP gene as housekeeping. For that, 1 µL of cDNA was amplified in a 10 µL PCR reaction containing 0.5 µL of each primer (500 nM), 5 µL of PowerUp SYBR Green Master Mix (Applied Biosystems) and 3 µL of RNase/DNase-free water. PCR began with a first cycle at 95 °C for 3 min followed by 55 cycles, each composed of a denaturation step , an annealing step  and an elongation step . The primers employed in the amplification were as follows: ZO-1 F, 5′-GGTGAAGTGAAGACAATG-3′; ZO-1 R, 5′-GGTAATATGGTGAAGTTAGAG-3′; occludin F: 5′-ATGAGACAGACTACACAACTGG-3′; occludin R: 5′-TTGTATTCATCAGCAGCAGC-3′; GADPH F: 5′-TGCACCACCAACTGCTTAGC-3′; GADPH R: 5′-GGCATGGACTGTGGTCATGAG-3′. A melting curve was employed to ensure the specificity of the amplification products. The mRNA levels of each protein in the different samples were normalized against GADPH as a housekeeping gene and expressed as the fold increase with respect to their respective control using the E−ΔΔCT method.Prior to analysis, wine was filtered through a 0.22 μm filter , and defrosted aliquots of IDW or CDW supernatants were filtered through a 0.22 μm filter . The UPLC-ESI-MS/MS method for the determination of phenolic compounds was described in a previous study . The mulPellets from the fecal fermentations in the absence and presence of IDW were subjected to DNA extraction and 16S rDNA sequencing. Initially, several microbial counts were realized. Then, DNA was purified using the QIAamp DNA Stool Mini Kit , following the manufacturer’s recommended protocol.® protocol was used to prepare the libraries, including PCR Blockers in the first process for minimizing the amplification of mitochondrial and cloroplast DNA [® MiSeq instrument  using 2 × 500 paired-end reads and then it was analyzed by amplifying and sequencing the V3-V4 region of the 16S rRNA gene. Raw files are available in the National Center for Biotechnology (NCBI) repository under project code PRJNA731070.The two-step PCR Illuminalast DNA . The V3-To denoise, align pairs and filter out chimeras in the raw data the DADA2 algorithm was employed . The errAliquots of CDW supernatants from the fecal fermentations in the absence and presence of IDW were defrosted and filtered through a pore size of 0.22 µm. Analysis of SCFAs was carried out following the SPME-GCMS method in accordance with the methodology described in a previous work . The SCFAll the statistical tests were carried out using R. Each treatment tested in Caco-2 cells was individually compared to the control incubation employing the T-test (vegan package) for both paracellular transport and TJ protein expression assays. The alpha diversity indices were calculated using the phyloseq package, and the “Heatplus“ and “gplots” packages were used to show the relative abundance of bacterial genera with more than 1% of abundance.v/v) of the two digested-wine fluids  at two incubation times (4 h for IDW and 16 h for CDW) was initially evaluated (results not shown). Cytotoxicity was associated with the components of the media used in the simulated digestions  rather than the phenolic compounds themselves. Finally, for both fluids, 1:40 (v/v) dilutions were selected for further assessment of their effects on paracellular permeability as lower dilutions were harmful (>40% cytotoxicity) to cells.The toxicity on Caco-2 cells of different dilutions  (p > 0.05) on paracellular permeability with respect to the control (p < 0.001) reduced paracellular transport of FITC-dextran (p > 0.05) were found (p = 0.0201) between the CDW and its corresponding medium (CDM), which indicated that the presence of wine-derived metabolites seemed to counteract the pro-leaky effects of the compounds present in the fermentation medium by itself. Additionally, the standard 3,4-dihydroxyphenylacetic acid  at 200 µM (or 33.6 mg/L) reduced the paracellular transport of FITC-dextran in a statistically significant way (p < 0.001)  . Note thcontrol) . At smal control a. Non-si-dextran a. In simre found b. Howeve< 0.001) b.v/v) to avoid cytotoxicity, the hypothetical total phenolic contents of the IDW- and CDW-diluted solutions in contact with cells were, respectively, 15.6 and 7.8 mg/L, which was in the concentration range used in other in vitro studies that found non-significant effects on paracellular permeability.Numerous studies have reported protective effects on the paracellular permeability of phenolic-rich products using different cell models under inflammatory or homeostatic conditions ,25,26,27Another fact that should be taken into consideration in interpreting the results is the presence of ethanol in wine. Ethanol has been reported to increase permeability in people who consume alcohol excessively on a daily basis . Wang etOur results with quercetin (200 µM) concurred with previous studies that reported how quercetin enhanced cellular integrity in cell models, either in the absence or presence of stressful conditions of some kind. Among others, Suzuki and Hara  found a p > 0.05)  and CDW (after 16 h of incubation) a,c. Howe > 0.05) b,d. The  > 0.05) a,b, wher > 0.05) c,d.For the intestinal barrier to work properly, it requires the presence of TJ, which are central components of the barrier structure. Several studies have reported that polyphenols can also improve the synthesis and redistribution of TJ proteins, although results are quite variable ,32,33,34Therefore, under the in vitro conditions used in this study, wine seemed not to notably affect paracellular permeability or TJ protein expression, which did not discard a direct potential protective role in the intestinal barrier of wine polyphenols as seen for quercetin and its microbial-derived metabolite, 3,4-dihydroxyphenylacetic acid.p-coumaroyl) glucoside. On the other hand, 3-O-methygallic acid was found in IDW but was not present in wine (v/v) dilution inherent to the simulated gastrointestinal digestion. In contrast, gallic acid and its ethyl ester suffered a drastic reduction in their contents  after gastrointestinal simulation. Other compounds that showed reductions higher than 1:4 with respect to wine concentrations were: caffeic acid, ferulic acid, piceid, catechin, epicatechin, procyanidin B3, quercetin and its 3-O-glucoside, myricetin and its 3-O-rhamnoside, and some anthocyanins. Notably, compounds such as coumaric acid and resveratrol seemed to be released during gastrointestinal digestion as they showed a certain increase in their IDW concentrations in comparison to those in wine (1:1.9 for coumaric acid and 1:3 for resveratrol) (The phenolic characterization of the digested-wine fluids was carried out as an approach to determine the metabolic transformations that wine polyphenols suffer during digestion  and that in wine . Phenolieratrol) .v/v) diluted prior to its colonic fermentation, the concentration of protocatechuic acid slightly decreased after simulated colonic digestion . In contrast, 3-O-methylgallic acid and 4-hydroxybenzoic acids seemed to be formed during colonic fermentation as they exhibited even higher CDW concentrations than those in the previous intestinal-digested fluid  (In relation to the colonic-digested wine (CDW), only 3-O-methylgallic acid, protocatechuic acid, and 4-hydroxybenzoic acid present in the previous intestinal-digested fluid (IDW) were found after simulated colonic fermentation . Bearingctively) . NotablyTherefore, these results confirmed certain transformations of wine polyphenols during their in vitro digestion that might condition their bioactivity in the intestinal environment. For instance, 3-O-methylgallic acid, which appeared in the IDW fluid, was considered a product of the O-methylation of gallic acid and ethyl gallate after , which cAs an indirect mechanism, wine polyphenols and/or their metabolites might specifically affect IP by means of modulating gut microbiota. Therefore, microbiome composition as well as microbial metabolic activity  during the wine colonic fermentations were determined .As a first overview, Bacteroidaceae, Ruminococcaceae, Prevotellaceae, and Lachnospiraceae were the predominant families (higher percentage of relative abundance) in the initial stage of the fermentations  [Desulfovibrio genus, to use them as electron donors for reduction of sulfate or other oxidized sulfur compounds to generate H2S [Overall, it should be noted that these results were obtained under static fermentation conditions which imply certain substrate depletion and might explain, for instance, the increase of is genus . On the egraders , as wello acids) ; and therate H2S . In thisp < 0.05) higher concentrations were found for propionic acid, the most abundant acid  could also be contributing to the protective effect observed against the growth of some pathogen-related groups, such as ll-known , as wellll-known , and valy effect ,60. Furty effect . TherefoThis paper contributes with new evidence to the knowledge concerning the effects of polyphenols on intestinal health, and in particular on intestinal barrier integrity. Many mechanistic studies have reported the molecular effects on the intestinal permeability of dietary polyphenols without considering the metabolism reactions that they undergo within the human body. Therefore, one of the main inputs of this study has been to test digested-wine fluids obtained by using a well-standardized digestion model. Compositional analysis of the wine-derived intestinal- and colonic-digested fluids  confirmed active phenolic metabolism in both intestinal and colonic phases, in agreement with what is reported in the literature. Using a Caco-2 cell model under homeostatic conditions, non-significant effects on the paracellular permeability of the digested-wine fluids were observed, which was attributed to the relatively low phenolic concentration in the solutions tested. As regards the effects on TJ protein expression, slight increases in mRNA levels of occludin were observed for both IDW and CDW fluids, although without statistical significance. However, the fact that quercetin (present in wine and in IDW) and 3,4-dihydroxyphenylacetic acid (present in CDW) did lead to significant decreases in paracellular permeability in vitro led to us deciding not to discard a direct potential protective effect of whole polyphenols (and/or their metabolites) on the intestinal barrier in vivo. Another input of this paper has been to study the impact of wine-digested fluids on gut microbiota composition and functionality as an indirect mechanism to improve intestinal permeability. Results indicate that wine digestion promotes changes in the relative abundance of some families/genera as well as in the microbial production of SCFAs, all of which might affect paracellular permeability. Overall, this paper reinforces (wine) polyphenols as a promising dietary strategy to improve gut functionality and the relevance of microbial-derived phenolic metabolites in intestinal homeostasis.